C. burnetii causes the zoonotic disease Q fever and resides within monocytes/macrophages manipulating innate immune response. C. burnetii requires de novo protein synthesis and a functional Type IVB Secretion System (T4BSS) to modulate host the eukaryotic transcription factor Nuclear Factor-?B (NF-?B) signaling an essential regulator of innate response. Multiple pathogensincludingC.burnetiidelivereukaryotic-typeAnk-containingeffectorstohijackNF-?B signaling.C.burnetiiNineMileisolateencodes5Ank-containingeffectors(AnkA,-C,-F,-G,and -K).Experimentsfoundthat1)AnkKandAnkCsignificantlycontributestoC.burnetii?sabilityto inhibit NF-?B dependent gene expression and 2) ectopic expression of AnkK or AnkC in HEK293/hTLR4-MD2-CD14cellsblocksaccumulationofNF-?Binthenucleus.Thecentralfocus of this proposal is to determine mechanistically how the C. burnetii effectors, AnkK and AnkC, modulate NF-?B to promote intracellular survival and replication. The central hypothesis by accomplishingthefollowingspecificaims:1.DeterminethecontributionofAnkKand-Cto C. burnetii?s virulence. The working hypothesis is that C. burnetii AnkK and AnkC promotes pathogen virulence by modulating NF-?B activation. To test this, we will generate complementation strains of C. burnetii NM II (NM II) ankK::Tn and ankC::Tn mutants and investigate in vitro growth rescue and p65 nuclear translocation for phenotype rescue in tissue culturecells.Toinvestigateinvivogrowthrescue,wewillemploytheSCIDmice/NMIIinfection model. Employing CYA-translocation assays, we will then test Type 4 secretion system requirement.Furthermore,wewillgenerate?dotA,?AnkK,and?AnkCgeneknockoutsandtheir complementationstrainsinvirulentC.burnetiiNMI(NMI)toinfectC57Bl/6Nmiceanddetermine the relative contribution of AnkK and AnkC to bacterial virulence in vivo. 2. Determine the mechanism of AnkK/AnkC-dependent NF-?B inhibition during C. burnetii infection. The workinghypothesisisC.burnetiiAnkKorAnkCinteractswithhosttargetsinthecanonicalNF-?B pathway or potentially sequesters p65 to block its nuclear translocation. We will deplete p65 in hostcellstoinvestigategrowthrescueofNMIIankK::TnandankC::Tnmutants.Wewillexamine expression, phosphorylation and degradation status of individual components of the canonical NF-?BpathwayinstableTHP-1celllinesexpressingeGFP-taggedAnkKandAnkC.Toidentify host-binding partner(s), pull down assays (GFP-trap) will be performed with THP-1 stable cells expressingeGFP-AnkKand?AnkC.TodetermineC.burnetiiAnkKorAnkCdrivenmodulationof hostp65-dependentgeneexpressionininfectedcells,wewillperformscRNA-seqandp65/ChIP- seqexperiments.